Novel composition

ABSTRACT

Novel vaccine compositions are provided comprising a hepatitis B antigen formulated with aluminium phosphate. The vaccine compositions may additionally contain an inactivated hepatitis A virus, aluminium hydroxide and formol. The combined hepatitis A and B vaccine formulations can, if desired, be administered to human subjects in a 2 dose regimen. Suitable formulations are illustrated.

[0001] Vaccines for the prophylaxis of hepatitis A and hepatitis B infections are well known. For example the vaccine Engerix-B (Trade Mark) from SmithKline Beecham Biologicals is used to prevent Hepatitis B. This vaccine comprises hepatitis B surface antigen (specifically the 226 amino acid S-antigen described in Harford et al. in Postgraduate Medical Journal, 1987, 63 (Suppl. 2), 65-70) and is formulated using aluminium hydroxide as adjuvant. The vaccine Havrix (Trade Mark), also from SmithKline Beecham Biologicals can be used to prevent hepatitis A infections and is also formulated with aluminium hydroxide as adjuvant. This vaccine comprises an attenuated strain of the HM-175 Hepatitis A virus inactivated with formol (formaldehyde); see Andre et al [Prog Med. Virol. 1990, vol 37; p72-95]. The vaccine Twinrix (Trade Mark) which is a combination of the above hepatitis A and hepatitis B antigens may be used to protect against hepatitis A and hepatitis B simultaneously.

[0002] European patent 0 339 667 (Chemo Sero) describes the general concept of combining a hepatitis A antigen and a hepatitis B antigen to make a combination vaccine. In that specification it is stated that the adjuvant which is used is not critical: it must only be capable of enhancing the immune activity to a desired extent and not cause any side-effects. It is stated that aluminium gel may be used, in particular aluminium hydroxide gel and aluminium phosphate gel.

[0003] PCT application WO 93/24148 (SmithKline Beecham) describes the preparation of vaccines comprising hepatitis B surface antigen in which aluminium phosphate is used as adjuvant. Multivalent combination vaccines which may optionally contain a hepatitis A antigen, are described. The use of formol is not disclosed.

[0004] European Patent Number 0 633 784 (SmithKline Beecham) describes novel vaccine formulations comprising a hepatitis B component, particularly hepatitis B surface antigen, in combination with aluminium phosphate, and 3 de-O-acylated monophosphoryl lipid A.

[0005] It has now been surprisingly found that vaccines comprising hepatitis B and/or hepatitis A antigens give exceptionally good results if the vaccine is formulated in a specific manner.

[0006] Using vaccine formulations according to the invention, it is possible to administer the vaccines in a 2 dose, rather than a 3 dose, regimen.

[0007] In a first aspect of the invention, there is provided an aqueous vaccine composition comprising hepatitis B surface antigen which is formulated with aluminium phosphate as adjuvant, the concentration of aluminium phosphate being selected such that there is a ratio of 0.015-0.1 mg aluminium phosphate per ug hepatitis B surface antigen.

[0008] Preferably the ratio is in the range 0.02 to 0.08 mg aluminium phosphate per μg HBsAg.

[0009] In a further aspect of the invention an inactivated hepatitis A virus (HAV) may optionally be added to the formulation of the invention, providing a combined hepatitis A plus B vaccine which may be administered in a 2 dose schedule.

[0010] The hepatitis A antigen is preferably the HM-175 strain used in the commercial product Havrix (SmithKline Beecham Biologicals).

[0011] The concentration of hepatitis A antigen in the vaccine formulation of the invention is preferably about 720-2880 EU units per ml. For the definition of EU units see Andre et al (1990) loc cit.

[0012] The compositions of the invention which comprise HAV may additionally comprise aluminium hydroxide, the total amount of aluminium hydroxide generally being 0.05-0.10 mg per ml.

[0013] The total amount of aluminium salt per 0.5 or 1 ml dose is normally in the range 0.4-1.0 mg.

[0014] In the vaccine composition of the invention it is advantageous to add formol (formaldehyde) such that the formol concentration is 10-200 μg per ml.

[0015] Preferably the formol concentration is about 20-160 μg per ml.

[0016] Normally the hepatitis B antigen will be hepatitis B surface antigen (HBsAg). The preparation of Hepatitis B surface antigen (HBsAg) is well documented. See for example, Harford et al in Develop. Biol. Standard 54, page 125 (1983), Gregg et al in Biotechnology, 5, page 479 (1987), EP-A-0 226 846, EP-A-0 299 108 and references therein.

[0017] As used herein the expression ‘Hepatitis B surface antigen’ or ‘HBsAg’ includes any HBsAg antigen or fragment thereof displaying the antigenicity of HBV surface antigen. It will be understood that in addition to the 226 amino acid sequence of the HBsAg S antigen (see Tiollais et al, Nature, 317, 489 (1985) and references therein) HBsAg as herein described may, if desired, contain all or part of a pre-S sequence as described in the above references and in EP-A-0 278 940. HBsAg as herein described can also refer to variants, for example the ‘escape mutant’ described in WO 91/14703. In a further aspect the HBsAg may comprise a protein described as SL* in European Patent Application Number 0 414 374, that is to say a protein, the amino acid sequence of which consists of parts of the amino acid sequence of the hepatitis B virus large (L) protein (ad or ay subtype), characterised in that the amino acid sequence of the protein consists of either:

[0018] (a) residues 12-52, followed by residues 133-145, followed by residues 175-400 of the said L protein; or

[0019] (b) residue 12, followed by residues 14-52, followed by residues 133-145, followed by residues 175-400 of the said L protein.

[0020] HBsAg may also refer to polypeptides described in EP 0 198 474 or EP 0 304 578.

[0021] Normally the HBsAg will be in particle form. It may comprise S protein alone or may be as composite particles, for example (L*,S) wherein L* is as defined above and S denotes the S-protein of hepatitis B surface antigen.

[0022] Preferably the HBsAg will be adsorbed on aluminium phosphate as described in WO93/24148.

[0023] Preferably the hepatitis B antigen is HBsAg S-antigen as used in the commercial product Engerix-B (SmithKline Beecham Biologicals).

[0024] The vaccine formulations of the present invention will contain an immunoprotective quantity of the antigens and may be prepared by conventional techniques. Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Md., U.S.A. 1978.

[0025] Encapsulation within liposomes is described, for example, by Fullerton, U.S. Pat. No. 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Pat. No. 4,372,945 and by Armor et al., U.S. Pat. No. 4,474,757.

[0026] The vaccine compositions of the invention are preferably administered in a 0, 6 month schedule, that is to say a first dose at 0 months and a second dose at 6 months.

[0027] The vaccine compositions of the present invention are especially appropriate for adults and are also appropriate for administration to adolescents and children.

[0028] The following examples illustrate but do not limit the invention.

EXAMPLES Example 1 Specific Formulations

[0029] Specific formulations within the scope of the invention include the following:

[0030] a) A vaccine composition for administration to adults which comprises:

[0031] 40 μg HBsAg

[0032] 1440 EU HAV

[0033] 0.85 mg Aluminium salt (0.8 mg aluminium phosphate plus 0.05 mg aluminium hydroxide)

[0034] 20 kg formol

[0035] in a 0.5 ml dose

[0036] b) A vaccine composition for administration to adults which comprises:

[0037] 40 μg HBsAg

[0038] 1440 EU HAV

[0039] 0.85 mg Aluminium salt (0.8 mg aluminium phosphate plus 0.05 mg aluminium hydroxide)

[0040] 20 μg formol

[0041] in a 11.0 ml dose

[0042] c) A vaccine composition for administration to adolescents and/or children which comprises:

[0043] 20 μg HBsAg

[0044] 720 EU HAV

[0045] 0.45 mg Aluminium salt (0.4 mg aluminium phosphate plus 0.05 mg aluminium hydroxide)

[0046] 80 μg formol

[0047] in a 1 ml dose

[0048] d) A vaccine composition for administration to adolescents and/or children which comprises:

[0049] 20 μg HBsAg

[0050] 720 EU HAV

[0051] 0.45 mg Aluminium salt (0.4 mg aluminium phosphate plus 0.05 mg aluminium hydroxide)

[0052] 80 μg formol

[0053] in a 0.5 ml dose

Example 2 Study ‘HAB 054’

[0054] 2.1 One group of 47 healthy adult individuals was vaccinated with a vaccine composition (1 ml volume) containing:

[0055] 1440 EU HAV antigen

[0056] 40 μg HBsAg (S-antigen)

[0057] 0.8 mg AlPO₄

[0058] 0.05 mg Aluminium hydroxide

[0059] 20 μg/ml formol.

[0060] This is abbreviated in the tables below to ‘1440/NF40’

[0061] 2.2 One group of 47 healthy adult individuals was vaccinated:

[0062] a) in one arm with a vaccine composition containing 40 kg of HBsAg formulated with 0.8 mg AIPO₄, 20 μg/ml formol (hereinafter this composition is abbreviated to ‘NF 40’) in a 1 ml dose; and

[0063] b) in the opposite arm with HAVRIX 1440 containing 1440 EU HAV antigen on 0.5 mg Aluminium hydroxide and containing 20 μg/ml formol in a 1 ml dose.

[0064] The immunization schedule for HAB 054 was 0,6 (i.e. doses administered at month 0 and month 6).

[0065] The Hepatitis B serological results at months 2,6 and 7 (seroconversion (SC), seroprotection (SP) and geometric mean titre (GMT)) were unexpectedly high as compared to historical results obtained in another study (HBV NF 021) in which volunteers of the same age group were included.

[0066] Study HBV NF 021 (see Table I) included three groups:

[0067] Group 1: Vaccinated with a formulation containing 40 μg of HBsAg on 0.5 μg AIPO₄ in 0.5 ml volume without formol, schedule 0, 6 months (this is abbreviated in the table to NF 40 μg, 0, 6M)

[0068] Group 2: Vaccinated with a formulation containing 20 μg of HBsAg on 0.5 kg AIPO₄ in 0.5 ml volume without formol, schedule 0, 6 months (this is abbreviated in the table to NF 20 μg, 0, 6M)

[0069] Group 3: Vaccinated with an Engerix B formulation (20 kg) using a classical 3 dose immunization schedule of 0, 1, 6 months (third row of data in Table 1)

[0070] Results

[0071] 1) In study HBV NF 021, Hepatitis B serological results at month 7 in groups 1 and 2 were not satisfactory as compared to group 3 (lower SC, SP and GMT+/−1/3 (500 to 600) of the levels obtained after three doses of Engerix-B (1500)).

[0072] 2) In study HAB 054, the titres obtained after 2 doses of vaccine were of the same order of magnitude as those that would be obtained following three doses of Engerix B. TABLE 1 HBV - NF 021 STUDY Serconversion % Seroprotection % Geometric Mean Titre Month 2 Month 7 Month 8 Month 2 Month 7 Month 8 Month 2 Month 7 Month 8 NF 40 μg 50 98 98 16 93 96 5 883 683 (0.6 M) NF 20 μg 42 93 98 6 91 89 3 727 510 (0.6 M) Engerix B 74 100 100 50 100 100 23 1579 1550 20 μg (0.1.6 M)

[0073] TABLE 2 HAB 054 study HAB 2-dose program in adults (0, 6month schedule) N = 94 Seroconversion (in %) Anti-HBs HAB 054 month 2 month 6 month 7 month 9 month 12 HAV 1440/NF40 75 85 100 100 100 NF 40 (Havrix 80 87 96 97 97 1440 in other arm) HAV 1440/NF40: contains 0.85 mg Al salts 0.8 Al PO₄ 20 μg formol in 1 ml 0.05 Al (OH)₃ NF 40: contains 0.8 mg Al PO₄ 20 μg formol in 1 ml

[0074] TABLE 3 HAB 054 study HAB 2-dose program in adults (0, 6 month schedule) N = 94 Seroprotection (in %) Anti-HBs HAB 054 month 2 month 6 month 7 month 9 month 12 HAV 1440/NF40 36 62 100 100 100 NF 40 (Havrix 33 57 96 97 90 1440 in other arm) HAV 1440/NF40 contains 0.85 mg Al salts 0.8 Al PO₄ 20 μg formol in 1 ml 0.05 Al (OH)₃ NF 40: contains 0.8 mg Al PO₄ 20 μg formol in 1 ml

[0075] TABLE 4 HAB 054 study HAB 2-dose program in adults (0, 6month schedule) N = 94 geometric mean titre - anti-HBs ANTI-HBs month 2 month 6 month 7 month 9 month 12 HAV 1440/NF40 8.5 19 2286 1006 632 NF 40 (Havrix 8.8 18 1865 1107 484 1440 in other arm) HAV 1440/NF40: contains 0.85 mg Al salts 0.8 Al PO₄ 20 μg formol in 1 ml 0.05 Al(OH)₃ NF 40: contains 0.8 mg Al PO₄ 20 μg formal in 1 ml

[0076] TABLE 5 HAB 054 study HAB 2-dose program in adults (0, 6 month schedule) N = 94 S+ % - HAV Day month month month month month HAB 054 15 1 6 7 9 12 HAV 1440/NF40 78 100 100 100 100 100 NF 40 (Havrix 92 98 96 96 100 100 1440 in other arm) HAV 1440/NF40 contains 0.85 mg Al salts 0.8 Al PO₄ 20 μg formol in 1 ml 0.05 Al (OH)₃ NF 40: contains 0.8 mg AlPO₄ 20 μg formol in 1 ml

[0077] TABLE 6 HAB 054 study HAB 2-dose program in adults (0, 6 month schedule) N = 94 Geometric mean titre - Anti-HAV day month ANTI-HAV 15 month 1 month 6 month 7 month 9 12 HAV 1440/NF40 337 803 324 10.393 7.408 4077 NF 40 (Havrix 312 722 275 5.748 4.376 2882 1440 in other arm) HAV 1440/NF40: contains 0.85 mg Al salts 0.8 Al PO₄ 20 μg formal in 1 ml 0.05 Al (OH)₃ NF 40: contains 0.8 mg Al PO₄ 20 μg formal in 1 ml

Example 3 Studies ‘HAB 063, HABO71 and HAB 075’

[0078] HAB 2 Dose Program in Adolescents Aged 11-18

[0079] 3.1: Study HAB 063

[0080] Vaccine Composition Comprises:

[0081] 720 HAV EU/20 μg HBs Ag

[0082] 0.25 mg Al Salt

[0083] 40 μg formol

[0084] In 0.5 ml dose

[0085] Results are shown in Table 7. TABLE 7 HAB 063 study HAB 2-dose program in adolescents aged 11-18 years (0, 6 month schedule) N = 52 Antibody PI (m1) PI (m2) PI (m6) PII(m7) Anti-HAV S+ % 100 96.2 96.2 100 GMT (EL. U/ml) 504 318 199 6874 Anti-HBs S+ % 51.9 75 94.2 98.1 Seroprotection rate % 30.8 28.8 71.2 98.1 GMT (mlU/ml) 11 7 19 4110

[0086] 3.2: Study HAB 071

[0087] Two groups were used in this study.

[0088] Vaccine Composition Group 1:

[0089] 720 HAV EU/20 μg HBs Ag

[0090] 0.425 mg Al Salt

[0091] 40 μg formol

[0092] In 0.5 ml dose and at a schedule of 0, 6 months

[0093] Vaccine Composition Group 2 (Twinrix™ Junior):

[0094] 360 HAV EU/10 μg HBsAg

[0095] 0,225 mg Al Salt

[0096] 40 μg formol

[0097] In 0.5 ml dose and at a schedule of 0, 1, 6 months TABLE 8 HAB 071 study High Dose HAB (Group 1) PI (m1) PI (M2) PI (m6) PI (m7) Schedule 0, 6 n = 50 n = 48 n = 49 n = 49 Anti-HAV^(antibodies) S+ % 100.0 100.0 100.0 100.0 GMT (EL. U/ml) 641 347 161 8151 Anti-HBs^(antibodies) S+ % 74.0 70.8 85.7 100.0 Seroprotoection rate % 40.0 27.1 57.1 100.0 GMT (EL. U/ml) 12 9 17 4212 Twinrix ™ Junior(Group 2) PI (m1) PI (M2) PI (M2) PI (m7) Schedule 0, 1, 6 n = 49 n = 48 n = 48 n = 48 Anti-HAV^(antibodies) S+ % 95.9 100.0 97.9 100.0 GMT (EL. U/ml) 336 793 233 6394 Anti-HBs^(antibodies) S+ % 61.2 97.9 100.0 100.0 Seroprotoection rate % 36.7 83.3 97.9 100.0 GMT (EL. U/ml) 12 36 202 6330

[0098] 3.3 Study HAB 075

[0099] Two groups were used in this study.

[0100] Vaccine Composition Group I (Twinrix™):

[0101] 720 HAV EU/20 kg HBs Ag

[0102] 0.45 mg Al Salt

[0103] 80 μg formol

[0104] In 1 ml dose and at a schedule of 0, 6 months

[0105] N=67

[0106] Vaccine Composition Group 2:

[0107] 1440 HAV EU/40 μg HBs Ag

[0108] 0.85 mg Al Salt

[0109] 80 μg formol

[0110] In 1 ml dose and at a schedule of 0, 6 months

[0111] N=55 TABLE 9 HAB 075 study Group PI(m1) PI(m2) PI(m6) PII(m7) 1 Anti-HAV S+ % 97.0 95.5 100.0 100.0 GMT 349 193 135 5646 Anti-HBs S+ % 62.7 74.6 95.5 100.0 SP rate % 22.4 32.8 61.2 100.0 GMT 6 6 13 3046 2 Anti-HAV S+ % 100.0 100.0 100.0 100.0 GMT 533 318 249 9565 Anti-HBs S+ % 67.3 85.5 94.5 100.0 SP rate % 32.7 41.8 72.7 100.0 GMT 9 9 26 3497

Example 4 Study ‘HAB 084’

[0112] HAB 2 Dose Program in Adolescents Aged 12-15

[0113] Two groups were used in this study.

[0114] Vaccine Composition Group 1 (Twinrix™):

[0115] 720 HAV EU/20 μg HBs Ag

[0116] 0.45 mg Al Salt

[0117] 80 μg formol

[0118] In 1 ml dose and at a schedule of 0, 6 months

[0119] Vaccine Composition Group 2 (Twinrix™ Junior):

[0120] 360 HAV EU/10 μg HBs Ag

[0121] 0.225 mg Al Salt

[0122] 40 μg formol

[0123] In 0.5 ml dose and at a schedule of 0, 1, 6 months TABLE 10 HAB 084 study Group 1 PI(m1) PI(m2) PI(m6) PII(m7) (0, 6) N = 142 N = 142 N = 142 N = 142 Anti-HAV S+ % 99.3 100 100 100 GMT 348 244 178 5486 Anti-HBs S+ % 80.5 81 93 100 SP rate % 43 38 68 97.9 GMT 14 9 20 4948 2 PI(m1) PII(m2) PII(m6) PIII(m7) (0, 1, 6) N = 148 N = 146 N = 147 N = 147 Anti-HAV S+ % 93.2 99.3 99.3 100 GMT 227 548 298 4174 Anti-HBs S+ % 58.1 95.9 99.3 100 SP rate % 29.1 85.6 98 100 GMT 9 42 305 5054

Example 5 Study ‘HAB 076’

[0124] HAB 2 Dose Program in Children Aged 1-11 Years

[0125] Vaccine Composition Comprises:

[0126] 720 HAV EU/20 μg HBs Ag

[0127] 0.45 mg Al Salt

[0128] 80 μg formol

[0129] In 1 ml dose at a schedule of 0, 1 month

[0130] Results are shown in Table 11. TABLE 11 HAB 076 study PI(m1) PI(m2) PI(m6) PII(m7) N = 194 N = 201 N = 197 N = 199 Anti-HAV S+ % 99.5 98.5 98 100 GMT 434 293 193 11543 Anti-HBs S+ % 72.7 89.1 93.9 100 SP rate % 30.3 47.3 78.2 98.5 GMT 8 11 34 8056

SUMMARY

[0131] The clinical results shown in the above examples clearly indicate that a satisfactory immune response is obtained, both for hepatitis A and hepatitis B after the full schedule, that is by Month 7, in children, adolescents and adults. 

1-18 (Cancelled).
 19. A method of inducing a seropositive antibody response against hepatitis A virus and hepatitis B virus and a seroprotective antibody response against hepatitis B virus in infants or adolescents, the method comprising administering two doses of a vaccine composition comprising hepatitis B surface antigen, an inactivated hepatitis A virus, formol and an adjuvant comprising one or more aluminum salts.
 20. The method of claim 19 wherein the vaccine composition comprises 10 to 200 micrograms per milliliter of formol.
 21. The method of claim 19 wherein the concentration of inactivated hepatitis A virus is about 720 to 2880 EU per milliliter.
 22. The method of claim 19 wherein the concentration of hepatitis B surface antigen is 20 to 40 micrograms per milliliter.
 23. The method of claim 19 wherein the inactivated hepatitis A virus if derived from the HM-175 strain.
 24. The method of claim 19 wherein the hepatitis B surface antigen is the S-antigen.
 25. The method of claim 19 wherein a second dose of the vaccine composition is administered 1 month after administration of a first dose.
 26. The method of claim 19 wherein a second dose of the vaccine composition is administered 6 months after administration of a first dose. 